Millions of Americans are affected by permanent hearing deficits and balance dysfunctions that result from losses of sensory hair cells. In mammalian ears, when hair cells die they are not effectively replaced, but the case is quite different in non-mammalian vertebrates. In those species hair cell loss leads to cell proliferation and the subsequent differentiation of new replacement hair cells, which then become innervated. These regenerative events lead to structural healing of damaged ears and can restore hearing and balance within one month after non-mammalian vertebrates have experienced types damage that would cause permanent deficits in our own ears. Research has shown that human and rodent ear tissues can activate the biological machinery that underlies regeneration, but the regenerative responses in mammalian ears are normally limited. We are seeking the means to overcome those limits. In this project we shall conduct experiments that are designed to identify the mechanisms that lead to hair cell differentiation and we are seeking to produce large numbers of hair cells in vitro. The controlled expression of transcription factors will be used to induce hair cell differentiation in vitro. In addition we shall seek to identify extracellular signals that will induce embryonic stem cells to differentiate otic phenotypes in vitro. To determine whether embryonic stem cells, transfected inner ear epithelial cells, and different lines of immortalized otic cells can be induced to differentiate as hair cells we will transplant them into ears developing in vivo and then assess the expression of otic markers. We also shall seek to identify culture conditions that promote the differentiation of cells dissociated from ears of embryonic mice and we shall develop methods for clonal expansion of otocyst-derived stem cells. The availability of lines of specialized cells that can be produced in vitro has provided the basis for significant gains in understanding cellular development, function, and pharmacology in cancer cell biology, lipid metabolism, diabetes, neurology, and other fields of biomedical science. Such cell lines and the knowledge gained in the development of lines of cells differentiated from embryonic stem cells are likely to play vital roles in the development of treatments for disease. By meeting the goals of this project we hope to contribute to the translation of cell biology into advances that will be useful for understanding and treating diseases of hearing and balance. [unreadable] [unreadable]